ARTICLE
Received 6 May 2016 | Accepted 23 Aug 2016 | Published 4 Oct 2016
DOI: 10.1038/ncomms13028
OPEN
Retinal transplantation of photoreceptors results
in donor–host cytoplasmic exchange
Tiago Santos-Ferreira1,*, Sı́lvia Llonch1,*, Oliver Borsch1,*, Kai Postel1, Jochen Haas1 & Marius Ader1
Pre-clinical studies provided evidence for successful photoreceptor cell replacement therapy.
Migration and integration of donor photoreceptors into the retina has been proposed as the
underlying mechanism for restored visual function. Here we reveal that donor photoreceptors
do not structurally integrate into the retinal tissue but instead reside between the photoreceptor layer and the retinal pigment epithelium, the so-called sub-retinal space, and
exchange intracellular material with host photoreceptors. By combining single-cell analysis,
Cre/lox technology and independent labelling of the cytoplasm and nucleus, we reliably track
allogeneic transplants demonstrating cellular content transfer between graft and host
photoreceptors without nuclear translocation. Our results contradict the common view that
transplanted photoreceptors migrate and integrate into the photoreceptor layer of recipients
and therefore imply a re-interpretation of previous photoreceptor transplantation studies.
Furthermore, the observed interaction of donor with host photoreceptors may represent an
unexpected mechanism for the treatment of blinding diseases in future cell therapy
approaches.
1 CRTD/Center
for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstrasse 105, 01307 Dresden, Germany. * These authors
contributed equally to the work. Correspondence and requests for materials should be addressed to M.A. (email: [email protected]).
NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13028
P
hotoreceptor transplantation has been shown to repair
retinal function in mouse models of retinal degeneration1–3.
Therefore, it is considered as a potential cell replacement
treatment option for retinopathies characterized by photoreceptor
loss such as retinitis pigmentosa and age-related macular
degeneration. Following sub-retinal, that is, between the
photoreceptor/outer nuclear layer (ONL) and retinal pigment
epithelium, transplantation of photoreceptor precursors into
partially degenerated adult retinas, several reports provided
evidence for successful migration, integration and maturation of
donor cells into the host’s ONL1–6.
In most transplantation studies, donor cells expressing a
fluorescent reporter are used to facilitate their identification
within the host tissue. However, sole reporter detection can be
misleading, as it has also been found in host cells due to donor–
host cell fusion7–9. Indeed, transplantation studies performed
more than a decade ago using Cre/LoxP technology and detection
of fluorescent bi-nucleated heterokaryons demonstrated
spontaneous fusion between bone marrow-derived donor
cells and diverse host cell types, including Purkinje neurons,
cardiomyocytes and hepatocytes9, thereby challenging the
hypothesis of somatic (stem) cell transdifferentiation into
multiple cell types10.
In previous photoreceptor transplantation studies, a potential
fusion between donor and host cells was ruled out, as fluorescent
reporter positive photoreceptors found within the ONL contained
just a single nucleus and, moreover, some fluorescently labelled
processes of donor cells showed no co-labelling when grafted into
hosts expressing a different fluorescent marker4,5. In general,
exchange of cytoplasmic content between cells is not restricted
to the generation of syn- or heterokaryons, that is, the
generation of a single cell containing two nuclei, but can also
be observed between morphologically distinct cells that form
membrane connections10 including plasma bridges/tunnelling
nanotubes11,12 or transfer intracellular material by vesicular
transport processes13.
In this study, we reinvestigated the potential occurrence of
fusion events following allogeneic photoreceptor transplantation
by (i) single-cell analysis using flow cytometry, (ii) the Cre/LoxP
fusion assay and (iii) separating cytoplasmic from nuclear
labelling. With these experiments we observe that, after retinal
transplantation, the majority of grafted photoreceptors do not
structurally integrate into recipient tissue but instead exchange
intracellular material with host photoreceptors.
Results
Double fluorescent photoreceptors after transplantation.
Initially, postnatal day (P) 4 photoreceptors, isolated from
reporter mice expressing cytoplasmic enhanced green fluorescent
protein (eGFP) driven by the rod-specific neural retina leucine
zipper promoter (Nrl-eGFP14), were transplanted into the subretinal space of recipient mice ubiquitously expressing DsRED15,
to assess whether single cells within host retinas contained both
reporters (Fig. 1a). Three weeks after grafting, flow cytometric
analysis identified cell populations within host retinas expressing
eGFP or DsRED, as well as a robust number of double-labelled
(eGFP þ /DsRED þ ) cells (98±17 cells per 1 million photoreceptors; n ¼ 3; Fig. 1b). Further analysis of individual experimental
retinas (n ¼ 3) by imaging flow cytometry, a technique allowing
concurrent visualization of individual events during flow
cytometry (Supplementary Fig. 1), revealed that 80% of doublepositive cells indeed expressed both fluorescent reporters, that is,
GFP and DsRED (Fig. 1c). In contrast, in control samples
containing a mixture of enriched P4 GFP þ photoreceptors with
DsRED þ retinal cells, double positivity was only observed due to
2
cell doublets or attached fluorescent debris (Fig. 1c). These results
suggest the exchange of cytoplasmic content or fusion between
donor and host photoreceptors.
Exchange of intracellular content between donor and host. To
directly assess the exchange of intracellular content between
donor and host cells, photoreceptors isolated from a conditional
mouse reporter line containing a loxP-flanked stop cassette
upstream of the tdTomato reporter gene (Ai9 mice16) were
transplanted into photoreceptor-specific Cre-recombinase
expressing hosts (B2-Cre þ / # ; n ¼ 6; Fig. 2a)17. Three weeks
after grafting, donor cells located in the sub-retinal space
(357±96 cells per retina; Fig. 2f) as well as photoreceptors
within the host ONL (443±103 cells per retina; Fig. 2f) showed
expression of tdTomato (Fig. 2b–e), whereas no reporter
expression was detected on transplantation of Ai9 donor cells
into B2-Cre # / # recipients (Fig. 2f and Supplementary Fig. 2).
This result indicates that Cre-recombinase produced by host
photoreceptors translocates into donor cells enabling the excision
of the stop cassette and thus expression of the reporter protein.
The detection of the fluorescent reporter also within
photoreceptors of the recipient’s ONL suggests a bidirectional
exchange of intracellular material between donor and host cells
or, alternatively, could be due to integration of fluorescentlabelled cells, following unidirectional transfer of Crerecombinase, into the ONL.
GFP þ cells in the host ONL lack a donor-derived nucleus. To
distinguish between potential integration of donor photoreceptors
and fusion events, donor cells with the nucleus and cytoplasm
independently labelled were used for grafting experiments.
Therefore, Nrl-eGFP mice were repeatedly injected during
development with the thymidine analogue 5-ethynyl-2́-deoxyuridine (EdU) to label nuclei of proliferating cells within the retina
(Supplementary Fig. 3a–d). Subsequently, P4 photoreceptor
precursors containing cytoplasmic GFP and EdU-labelled nuclei
were sub-retinally transplanted into adult wild-type mice. Three
weeks after transplantation, the ONL of experimental retinas
(n ¼ 3) contained GFP-labelled cells with mature photoreceptor
morphology (Supplementary Fig. 3e) located next to clusters of
GFP þ grafts residing in the sub-retinal space. Although the
majority of GFP þ cells placed in the sub-retinal space were also
EdU þ (Supplementary Fig. 3e–h), no EdU labelling was observed
in GFP þ cells located within the ONL (Supplementary
Fig. 3e,i–k). These results suggest that integration of donor cells
into the host ONL does not occur or is a rare event; instead,
cytoplasmic content, but not the entire cell nuclei/nuclear
genomic DNA, might be exchanged from donor cells to host
photoreceptors.
As consecutive EdU injections during development did
not label all photoreceptors of the P4 retina (Supplementary
Fig. 3b–d), Y-chromosome fluorescent in situ hybridization
(FISH) was used to identify all nuclei within the donor cell
fraction (Fig. 3 and Supplementary Fig. 4), allowing quantitative
analysis. Therefore, donor photoreceptors were isolated from
male Nrl-eGFP mice and transplanted into female wild-type
recipients (Fig. 3a). After 3 weeks, virtually all analysed GFP þ
cells within the sub-retinal space contained a Y-chromosome
identifying them as donor photoreceptors (98.24±0.77%;
Fig. 3b–f,k). In contrast, of all GFP þ photoreceptors located
within the host ONL, only few cells showed co-staining for the
Y-chromosome (1.2±0.42%; Fig. 3b,g–j, k, l–o), confirming that
cytoplasmic content but not the nucleus of donor cells is
translocated into host photoreceptors.
NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13028
a
Retinal transplantation
Nrl-eGFP
DsRED
Donor
photoreceptor
Conventional
flow cytometry
Host cells
Imaging
flow cytometry
Control
“Fused”
photoreceptor
Nrl-eGFP
+
Nrl-eGFP into DsRED
GFP
0.001
85.2
14.8
Nrl-eGFP
DsRED
0.01
0
0
25.7
74.3
92.8
0
7.2
0
eGFP+/DsRED+ photoreceptors
per 1 million cells
b
Nrl-eGFP into DsRED
n=3
150
100
50
0
DsRED
c
Nrl-eGFP into DsRED
GFP
DsRED
BF
+
GFP
+
DsRED
Percentage of cells in
double positive gate
BF
GFP
+
DsRED
n=3
100
80
60
40
20
0
Double
positive
False
double
positive
Control: Nrl-eGFP/DsRED mix
n=3
GFP
Percentage of cells in
double positive gate
100
100%
80
60
40
20
0
0%
Double
positive
False
double
positive
DsRED
Figure 1 | Analysis of DsRED host retinas grafted with Nrl-eGFP photoreceptors by conventional and imaging flow cytometry. (a) Schematic
representation of experimental outline: transplantation of P4 eGFP þ photoreceptors into the retina of adult DsRED hosts and corresponding control
(mixing of enriched P4 eGFP þ photoreceptors with DsRED þ retinal cells) with subsequent conventional and imaging flow cytometry analysis. (b) Flow
cytometric analysis of individual experimental retinas (n ¼ 3) revealed a robust number of GFP þ /DsRED þ double-labelled photoreceptors (98±17
photoreceptors per 1 million photoreceptors) within DsRED host retinas 3 weeks after transplantation of eGFP þ donor cells (b, left plot), indicating
exchange of cytoplasmic material between donor and host photoreceptors. Nrl-eGFP (b, right plot) or DsRED (b, middle plot) retinas do not contain
double-positive cells. (c) In transplanted retinas (Nrl-eGFP into DsRED; n ¼ 3), imaging flow cytometry identified single GFP þ /DsRED þ cells (three
exemplary cells are shown) in the double-positive cell population (upper left plot, blue gate). The majority of cells (B80%; upper right graph) showed
double fused fluorescence, whereas B20% were false double-positive (that is, containing doublets or attached fluorescence debris). All double-positive
events in Nrl-eGFP mixed DsRED controls (lower left plot, blue gate; lower right graph; n ¼ 3) were identified as false double-positive (100%; three
exemplary cells are shown). Scale bar, 5 mm. Data represent mean±s.e.m. BF, bright field.
NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13028
a
CAG Pr
xP
xP
Lo
Lo
Stop
tdTomato
+/–
B2-Cre
RPE
SRS
OS/IS
ONL
3 Weeks
Ai9
Donor photoreceptor
precursor
“Fused” donor photoreceptor
“Fused” host photoreceptor
B2-Cre+/– host photoreceptor
tdTomato / DAPI
b
c
SRS
SRS
ONL
ONL
d
e
f
Number of tdTomato+
cells/retina
800
ONL
B2-Cre+/–
B2-Cre–/–
600
400
200
0
ONL
L
ON
al
tin
bre
u
S
L
ON
al
tin
bre
u
S
Figure 2 | Visualization of fusion events following photoreceptor transplantation using Cre/LoxP technology. (a) Schematic representation of
experimental outline to visualize transfer of cytoplasmic material between donor and host cells by Cre/LoxP recombination. (b–e) Donor photoreceptors
isolated from floxed reporter mice (Ai9) and transplanted into rod photoreceptor-specific Cre mice (B2-Cre þ / # ) show expression of the tdTomato
reporter in sub-retinal (SRS) located cells (b–e; arrow head in d,e) revealing transfer of Cre-recombinase from host photoreceptors to donor cells. tdTomato
is also present in photoreceptors located in the ONL (b–e; arrow in d,e), suggesting bidirectional transport of intracellular content between donor and host
photoreceptors. (f) Quantification of tdTomato þ cells in the sub-retinal space and in the ONL of B2-Cre þ / # hosts (n ¼ 6) show similar amounts of
reporter-labelled cells (ONL: 443±103 cells per retina; SRS: 357±96 cells per retina). No tdTomato-expressing cells were detected in transplanted
B2-Cre # / # mice (n ¼ 4). Data represent mean±s.e.m. Scale bar,: 20 mm (c,e). CAGS Pr, CAGS promoter; INL, inner nuclear layer; IS/OS, inner/outer
segments; ONL, outer nuclear layer; RPE, retinal pigment epithelium; SRS, sub-retinal space.
Discussion
Photoreceptor replacement therapies have shown promising
results leading to functional restoration of rod and cone-mediated
vision1,3. Migration and integration of grafted photoreceptors
into the host’s retinal circuitry has been hypothesized as the
underlying mechanism for vision improvement18. However, our
results reveal that de novo integration of photoreceptor
transplants is a rare event. Conversely, sub-retinally located
donor cells exchange cytoplasmic content with endogenous
photoreceptors. Thus, potential restoration of visual function
might have been achieved by material transfer, suggesting a new
powerful mechanism to rescue dysfunctional and degenerating
photoreceptors. Our findings will have strong implications for a
re-evaluation of the field of photoreceptor transplantation.
Interestingly, reporter expression within the host retinal tissue
was only observed in the ONL and exclusively detected in
photoreceptors, suggesting that exchange of intracellular content
is restricted to photoreceptor–photoreceptor interactions. Indeed,
transplantation of other fluorescent retinal cell types, for example,
retinal progenitors or CD73 # cells, resulted only in very few
reporter-labelled photoreceptors in the ONL4,6,19. In addition,
4
recent data on transplantation of photoreceptors into mouse
models of severe retinal degeneration with complete loss of
photoreceptors did not result in cells within the recipient’s retinal
tissue with reporter labelling20,21, suggesting that retinal cells of
the inner nuclear layer, that is, horizontals, bipolars, Müller glia
or amacrines, do not exchange cytoplasmic content with donor
photoreceptors. Notably, grafted photoreceptors in severely
degenerated retinas still showed signs of a mature phenotype
including expression of phototransduction proteins, outer
segment-like structures and synapse formation that resulted in
some functional improvements20,21. Therefore, disease states
characterized by complete loss of photoreceptors represent a
promising target for photoreceptor replacement approaches in
future clinical applications.
Previous studies reported fluorophore-labelled cells within the
host ONL, not only in wild-type hosts but also in several genetic
degeneration mouse models that contain dysfunctional photoreceptors1–4. Further studies need to be performed, to evaluate
whether donor–host transfer of cytoplasmic content occurs in the
diseased retina as well. Interestingly, the described morphology of
fluorophore-labelled cells within the ONL strongly correlated
NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13028
a
Nrl-eGFP
3 Weeks
WT
RPE
SRS
OS/IS
“Fused” host photoreceptor
with grafted cell
Male photoreceptor
ONL
Female photoreceptor
Integrated photoreceptor
Y-Chromosome/Nrl-eGFP/DAPI
b
k
c–f
g–j
ONL
Host retina
%Y-chromosome+/GFP+
cells in the host retina
SRS
105
100
95
3
0
SRS
c
d
e
f
SRS
SRS
SRS
SRS
g
h
ONL
ONL
ONL
ONL
m
n
o
ONL
ONL
ONL
l
ONL
i
L
ON
j
Figure 3 | Donor cell-derived eGFP but no nuclei are located in host photoreceptors. (a) Experimental overview: Nrl-eGFP male photoreceptor
precursors were transplanted into wild-type female retinas. Sections of individual experimental retinas (n ¼ 5) were analysed by Y-chromosome FISH and
immunohistochemistry for eGFP. (b) Overview of grafted area 3 weeks after transplantation (increased space between graft and host ONL results from a
histological (cutting) artefact). (c–f) Y-chromosome þ /GFP þ cells were located in the SRS, identifying them as donor photoreceptors (see also arrowhead
in g–j). (g–j) Y-chromosome # /GFP þ cells were detected in the ONL of the host retina (arrows), demonstrating transfer of donor cytoplasmic content but
not nuclei to host photoreceptors. (k) Quantification of GFP þ cells containing Y-chromosome in the SRS and in the ONL of the host. Although the vast
majority of the GFP þ cells in the SRS contained a Y-chromosome (98.24±0.77%), only 1.2±0.42% of GFP þ cells in the ONL showed a positive staining
for the Y-chromosome. Data represent mean±s.e.m. (l–o) Very few GFP þ cells within the ONL of the host retina were also Y-chromosome þ , indicating a
low extent of structural integration of donor cells into the host retina. Scale bars, 20 mm (f,i,j,o). INL, inner nuclear layer; IS/OS, inner/outer segments; ONL,
outer nuclear layer; RPE, retinal pigment epithelium; SRS, sub-retinal space.
with the morphology of host photoreceptors in different
degeneration models2, suggesting a potential fusion of donor
and host photoreceptors also in a disease environment. Besides
the presence of fluorescent reporters, correct expression of
proteins missing in the respective disease models was observed
in ONL located photoreceptors and even functional recovery was
NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13028
achieved
following
photoreceptor
transplantation
in
dysfunctional, mildly degenerated retinas1–3. Based on our
findings, we conclude that the majority of donor cells remain in
the sub-retinal space and thus may not form direct synaptic
connections to endogenous secondary neurons. Hence, we
hypothesize that the observed visual improvements1–3 might
result from remaining endogenous photoreceptors supplemented
by donor cell-derived proteins. Interestingly, reporter labelled
photoreceptors within the ONL have been observed up to 6
months following transplantation, although in significantly lower
numbers than after 4 weeks2,22, implying stable photoreceptor–
photoreceptor connections or, alternatively, dynamic re-arrangements of cell–cell contacts over long periods of time given that
GFP has a half-life of B26 h23. Improving fusion efficiency, as
reported for transplanted bone marrow cells with Purkinje
neurons in conditions such as inflammation, radiation
exposure, chemotherapeutic drugs, age or damage24 and
increasing long-time survival of transplanted photoreceptors
might therefore represent a new opportunity for cell-based
treatment approaches for photoreceptor degenerative diseases.
Interestingly, a recent study provided evidence that damageinduced fusion of haematopoietic stem and progenitor cells with
retinal ganglion or amacrine cells contributed to retinal tissue
repair25.
In conclusion, photoreceptor transplantation into host retinas
with remaining photoreceptors results in photoreceptor–
photoreceptor biomaterial exchange rather than migration and
integration into the recipient’s ONL. Transfer of cellular content
between donor and host cells has to be taken into account when
interpreting experiments using photoreceptor transplantation
into partially degenerated retinas with the intention to achieve
cell replacement. In fact, the potential exchange of cellular
material in any transplantation setting should be rigorously
evaluated given the increasing number of reports providing
evidence for complete9,25,26 or partial (our study) cell–cell fusion
events. Such knowledge will be an important prerequisite to
determine the impact of donor cells in tissue repair27. Although
the mechanism of vision improvement by photoreceptor
transplantation may be different than originally conceptualized,
capitalizing on the fusion-mediated exchange of cytoplasmic
components may represent a productive direction for the
development of cell support therapies.
Methods
Photoreceptor transplantation into the mouse retina. Photoreceptors were
isolated from postnatal day (P) 4 Nrl-eGFP14 or Ai9 (ref. 16; Gt(ROSA)
26Sortm9(CAG-tdTomato)Hze, Jackson Laboratory, Bar Harbor, USA) mice, enriched
by CD73-based magnetic-activated cell sorting and transplanted into the sub-retinal
space of adult (6–9 week) mice28. Therefore, eyes from donor P4 pups were
enucleated, rinsed in PBS and transferred to Hank’s balance salt solution (Gibco).
Next, retinas were isolated, digested to a single cell suspension using papain
dissociation (Worhington Biochemical Corporation) and the resulting cell suspension
was incubated with rat anti-mouse CD73 antibody (0.5 mg ml # 1 stock solution
diluted 1:40, BD Biosciences, ref: 550738) followed by anti-rat IgG Microbeads
(1:5 of stock solution, Miltenyi, Germany, ref: 130–048–502). Cell suspensions were
rinsed, spun down, ran through LS Columns attached to a magnet and CD73 þ cell
fractions (rod photoreceptors) were concentrated by centrifugation and resuspension
to a 2 $ 105 cell per ml solution. Using a blunt 34 Gauge needle attached to a 10 ml
Hamilton syringe, donor cells were trans-vitreally transplanted into the sub-retinal
space of recipients, that is, wild-type, ubiquitous DsRED (actin-DsRED, (15),
B6.Cg-Tg(CAG-DsRed*MST)1Nagy/j, Jackson Laboratory) or rod photoreceptorspecific B2-Cre17 mice. All animal experiments were approved by the ethics
committee of the TU Dresden and the Landesdirektion Dresden (approval number
24-9168.11-1/2008-33, 24-9168.11-1/2012-33 and 24-9168.11-1/2013-23) and
performed in accordance with the regulation of the European Union, German laws
(Tierschutzgesetz), the ARVO statement for the Use of Animals in Ophthalmic and
Vision Research, as well as the NIH Guide for the care and use of laboratory work.
EdU labelling of donor photoreceptors. The thymidine analogue EdU (20 mM;
Click-iT EdU Alexa Fluor 647 Imaging Kit; Invitrogen) was intraperitoneally
6
injected (1 ml per gram body weight) into female time-mated Nrl-eGFP mice 18
and 20 days after fertilization. Delivered pups received additional intraperitoneal
EdU injections (5 mM, 4 ml per gram body weight) at P1 and P3 before retinal
tissue was harvested at P4 for transplantation (see above) or histological analysis.
Prior immunohistochemistry (see below) EdU was detected on 20 mm-thick retinal
cryosections by following the manufacturer’s instructions using AlexaFluor 647
azide for visualization.
Immunohistochemistry. Transplanted mice were killed 3 weeks after transplantation, eyes enucleated and fixed in 4% paraformaldehyde (Merck Millipore)
for 1 h at 4 !C. The posterior part of the eye (sclera, choroid, retinal pigmented
epithelium and retina) was cryopreserved overnight at 4 !C in a 30%
(weight/volume) sucrose solution and embedded in optimal cutting medium
(OCT, NEG50, Thermo Scientific). Twenty-micrometre-thick sections were
collected on star frost slides, air-dried for 1 h at room temperature (RT), hydrated
with PBS for 30 min and incubated with blocking solution containing 0.3%
Triton-X100 (SERVA), 1% BSA (SERVA) and 5% donkey serum for 1 h at RT.
Slides were incubated with primary antibody against GFP (10 mg ml # 1 stock
solution diluted 1:1,000, AbCam, ref: AB13970) overnight at 4 !C, washed 3 times
for 10 min and respective Cy2-conjugated secondary antibody (1.5 mg ml # 1 stock
solution diluted 1:1,000, Jackson Immunoresearch, ref: 703225155) was added for
90 min in a 40 ,6-diamidino-2-phenylindole solution (DAPI, 1:20,000, Sigma).
Following incubation, slides were washed three times for 10 min in PBS and
mounted in AquaPolymount (Polysciences).
Image acquisition and processing. Fluorescent images were acquired using a
structured illumination microscope (Apotome ImagerZ1, Zeiss) and processed
offline using Fiji, Adobe Photoshop and Adobe Illustrator. Adobe Illustrator was
used to generate illustrations and schematic representations. Mouse illustration
used for schematic representations was simple mouse (c)Seans Potato Business,
CC-BY-SA-3.0.
FISH for Y-chromosome detection. For combined chromosomal FISH
(Y-chromosome FISH) and immunohistochemistry29, experimental retinas were
collected, fixed for 1 h at 4 !C with freshly prepared 4% paraformaldehyde
(Merck Millipore) and cryopreserved in 30% sucrose overnight at 4 !C.
Cryosections of 10 mm thickness were rehydrated with 10 mM citrate buffer and
treated with 2 $ saline sodium citrate buffer (SSC). Following pretreatment with
50% formamide for 1 h at RT, sections were washed in PBS for 5 min and incubated
with primary antibody against eGFP (10 mg ml # 1 stock solution diluted 1:800,
AbCam, ref: AB13970) overnight at 4 !C, followed by incubation with secondary
antibody conjugated to AlexaFluor 488 (1.5 mg ml # 1 stock solution diluted
1:1,000, Jackson Immunoresearch, ref: 103545155) for 90 min at RT. Next,
hybridization of the XMP Y orange probe (undiluted, Metasystems, ref:
D-1421-050-OR) to the Y-chromosome was performed. After loading the probe
into the hybridization chamber, those were sealed with rubber cement. To allow the
probe to penetrate the tissue, samples were incubated for 3 h at 45 !C. Then,
samples were transferred to a hot block at 80 !C for 5 min, to denature DNA.
Afterwards, probes were hybridized with DNA for 2 days at 37 !C.
Posthybridization consisted of 3 $ 15 min washes with 2 $ SCC at 37 !C and
2 $ 5 min stringency washes with 0.1 $ SCC at 60 !C. Finally, sections were
counterstained with DAPI (1:15,000, Sigma).
Quantification of fluorescent cells in retinal sections. Fluorescent labelled cells,
that is, eGFP, Ai9 (tdTomato) or Y-chromosome, were quantified on DAPI-stained
serial sections of experimental retinas (Z5 biological replicates) using fluorescence
microscopy (Apotome ImagerZ1, Zeiss). Respective data were plotted using
GraphPad Prism version 6 (GraphPad Software).
Flow cytometry. Transplanted retinas (n ¼ 3) and retinas from the used reporter
strains, that is, Nrl-eGFP (n ¼ 3) and DsRED (n ¼ 3), were dissociated to a singlecell suspension using papain dissociation (Worthington Biochemical Corporation)
for 20 min with 100 mg ml # 1 papain according to the manufacturer’s instructions,
followed by filtration through 30 mm cell strainers (Sysmex Partec). Samples were
analysed for reporter fluorescence, that is, eGFP and DsRED, with a BD FACSCanto II flow cytometer (BD Bioscience) after gating retinal cells for singlets
(FSC-H versus FSC-A and SSC-H versus SSC-A) and photoreceptors. Flow
cytometry data were processed offline using FlowJo software (Tree Star).
Imaging flow cytometry. For imaging flow cytometry, individual transplanted
retinas (n ¼ 3) were dissociated and processed as for conventional flow cytometry
(see above). Control samples (that is, mixed eGFP and DsRED cells) were generated as follows: P4 Nrl-eGFP retinas and adult DsRED retinas were separately
dissociated into a single-cell suspension and Nrl-eGFP rods were enriched using
CD73-based magnetic-activated cell sorting in line with the procedure for transplantation (see above). Each individual DsRED retina (n ¼ 3) was then mixed with
2 $ 105 Nrl-eGFP rods and spun down for further processing. Transplantation and
NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13028
control samples were analysed for reporter fluorescence (that is, eGFP and DsRED)
using an ImageStream X Mark II imaging flow cytometer (Amnis), after gating
retinal cells for singlets (aspect ratio versus area). For reasons of technical feasibility
(limitation of data volume per data set per sample), the majority of mere DsREDpositive cells was gated out and hence excluded from data acquisition by using
GFP fluorescence as a cutoff parameter. Data were processed offline using
IDEAS software (Amnis). Acquired individual images of double-positive
(GFP þ /DsRed þ ) cells (73, 34 and 31 individual cells from transplanted retinas;
12, 5 and 4 individual cells from the mixed control samples) were analysed and
quantified for the appearance of GFP and DsRED within the same cell, doublets
and cells with attached fluorescent debris.
Data availability. The authors declare that all data supporting the findings of this
study are available within the article and its Supplementary Information files or
from the corresponding author upon reasonable request.
References
1. Pearson, R. A. et al. Restoration of vision after transplantation of
photoreceptors. Nature 485, 99–103 (2012).
2. Barber, A. C. et al. Repair of the degenerate retina by photoreceptor
transplantation. Proc. Natl Acad. Sci. USA 110, 354–359 (2013).
3. Santos-Ferreira, T. et al. Daylight vision repair by cell transplantation.
Stem Cells 33, 79–90 (2015).
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Acknowledgements
We thank Sindy Böhme, Sabrina Richter and Katrin Sippel for animal husbandry,
Drs Yun Z. Le and Mike O. Karl for providing B2-Cre mice, Drs Anand Swaroop and
Sandra Cottet for providing Nrl-eGFP mice, and Drs Elly Tanaka, Gerd Kempermann,
Mike O. Karl and Annette E. Rünker for advice and critically reading the manuscript.
This work was supported by the Flow Cytometry Facility, a core facility of
BIOTEC/CRTD at Technische Universität Dresden. This work was financially supported
by the Deutsche Forschungsgemeinschaft (DFG) FZT 111 Center for Regenerative
Therapies Dresden, Cluster of Excellence (M.A.), DFG Grant AD375/3-1 (M.A.) and the
ProRetina Stiftung (M.A.).
Author contributions
T.S.F., S.L. and O.B. contributed to the experimental design, data acquisition, data
analysis and manuscript preparation. J.H. contributed to data acquisition. K.P.
contributed to experimental design and data acquisition. M.A. contributed to
experimental design, data analysis and manuscript preparation.
Additional information
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How to cite this article: Santos-Ferreira, T. et al. Retinal transplantation of photoreceptors results in donor–host cytoplasmic exchange. Nat. Commun. 7, 13028
doi: 10.1038/ncomms13028 (2016).
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NATURE COMMUNICATIONS | 7:13028 | DOI: 10.1038/ncomms13028 | www.nature.com/naturecommunications
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